We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.

Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

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Abstract. Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca 2+ ), phosphate (PO4 3-) and lactate. In all media containing no Ca 2+ , including medium lacking Ca 2+ , lacking Ca 2+ and PO4 3-, lacking Ca 2+ and lactate and lacking Ca 2+ , PO4 3-and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca 2+ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca 2+ .In conclusion, preincubation of thawed sperm in medium containing no Ca 2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca 2+ is practical for use in cryopreserved C57BL/6J sperm. Key words: C57BL/6, Calcium, Cryopreservation, In vitro fertilization, Sperm (J. Reprod. Dev. 55: [386][387][388][389][390][391][392] 2009) ryopreservation of mouse sperm is useful because it is simple, rapid and inexpensive. A large number of sperm can be frozen immediately after collection from a male. Females are required only when the frozen sperm are thawed for fertilization. Successful cryopreservation of mouse sperm was reported in 1990 using raffinose with glycerol [1], ME2SO [2] or skim milk [3]. Since then, cryoprotectant solutions and freezing methods for mouse sperm have been investigated [4][5][6][7][8][9][10][11]. At present, the 18% raffinose plus 3% skim milk method [12] is relied upon in most laboratories. However, with this technique, the fertility of cryopreserved sperm is far less than that of fresh sperm, especially with inbred mice [13][14][15][16]. In particular, fertility decreases markedly in C57BL/6J, a principal inbred strain used for transgenesis studies. Frozen C57BL/6J sperm exhibit acrosome damage with loss of contents caused by the freeze-thaw procedure [17], and almost no sperm can penetrate the zona pellucida. When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23]. Successful IVF with fresh sperm depends on the media used. Various IVF media have been developed, and the effects of media on capacitation and IVF have been investigated. However, the effects of media on frozen mouse sperm have not been investigated.In this s...